Efficacy of Saccharomyces yeast postbiotics on cell turnover, immune responses, and oxidative stress in the jejunal mucosa of young pigs

This study aimed to determine the effects of Saccharomyces yeast postbiotics on cell turnover, immune responses, and oxidative stress in the jejunal mucosa of pigs. Thirty-two newly weaned pigs at 6.05 ± 0.24 kg were assigned to two dietary treatments based on a randomized complete block design. The treatments were control group receiving a basal diet and a group supplemented with Saccharomyces yeast postbiotics (175 g/ton diet) in the basal diet. After 35 d of the study, pigs were euthanized and jejunal mucosa were collected to assess immune status, oxidative stress, barrier markers, cell proliferation, and apoptosis. Saccharomyces yeast postbiotics reduced (P < 0.05) the fecal score from d 3 to d 7 and tended to increase the gene expression of interferon-γ (IFN-γ) (P = 0.071) and mammalian/mechanistic target of rapamycin (mTOR) (P = 0.080), decrease the gene expression of B-cell lymphoma 2-associated X protein 1 (BAX1) (P < 0.05), tended to decrease the gene expression of serum and glucocorticoid-induced protein kinase 1 (SGK1) (P = 0.066), increased (P < 0.05) cell proliferation in the crypts, and tended to increase the villus height (P = 0.078) and crypt depth (P = 0.052) in the jejunum. In conclusion, the supplementation of Saccharomyces yeast postbiotics in nursery diets reduced diarrhea within the first week after weaning and provided protection to the villi in the jejunum by enhancing the immune responses of nursery pigs, promoting crypt cell proliferation, and reducing the expression of genes associated with apoptosis without affecting inflammatory and oxidative stress status in the jejunum of the nursery pigs.

Table 1.Fecal score of pigs fed diets supplemented with Saccharomyces yeast postbiotics. 1 Mean ± Standard error (n = 16). 2 Fecal scores greater than 5 were considered as severe diarrhea.

Gene expression of intestinal markers
The dietary treatments did not affect the Ct of the housekeeping gene β-actin (data not shown).The supplementation of Saccharomyces yeast postbiotics did not affect the gene expression of markers associated with intestinal barrier functions (Table 3).The supplementation of Saccharomyces yeast postbiotics tended to increase the gene expression of interferon-γ (IFN-γ) by 35% from 1.08 to 1.46 (P = 0.071), and mTOR by 38% from 1.01 to 1.39 (P = 0.080) in the jejunal mucosa of nursery pigs.The supplementation of Saccharomyces yeast postbiotics tended to decrease the gene expression of Serum and glucocorticoid-induced protein kinase 1 (SGK1) by 37% from 1.08 to 0.68 (P = 0.066) and reduced (P < 0.05) the gene expression of B-cell lymphoma 2-associated X protein 1 (BAX1) by 19% from 1.01 to 0.82 in the jejunal mucosa of nursery pigs.

Jejunal morphology and crypt cell proliferation
The supplementation of Saccharomyces yeast postbiotics tended to increase the villus height by 12% from 438 to 492 µm (P = 0.078) and the crypt depth (P = 0.052) by 10% from 246 to 270 µm in the jejunum (Table 4).The supplementation of Saccharomyces yeast postbiotics increased (P < 0.05) the cell proliferation in crypts of the jejunum by 14% from 53.3 to 60.6 units/crypt (as measured by immunohistochemistry identifying the Ki-67 + cells counted in a crypt).However, the supplementation of Saccharomyces yeast postbiotics in nursery pig diets did not affect the villus width, and the villus height to crypt depth ratio (VH:CD).

Discussion
A healthy intestinal mucosa plays a fundamental role in efficient nutrient absorption and prevents increased intestinal permeability 2 .In addition, the of intestinal mucosa is the first line of defense against pathogens invasion and its integrity is an essential factor for the maintenance of intestinal functions, including immune responses 8,28,29 .Along the gastrointestinal tract, the jejunum can be considered a key portion of analyzing the interaction among dietary compounds, microbiota, and the immune system 15 .
In this this study, the fecal score and the incidence of severe diarrhea were recorded to evaluate the overall health status of the pigs during the experimental period.Fecal score is a parameter largely used to evaluate the digestive health in animal models 9,[30][31][32][33] .In pig production, weaning is a stressful event that makes pigs more susceptible to diarrhea incidence, mainly due to the disruption of the intestinal microbiota 34 .Studies have reported that the effects of pathogenic infection, including enterotoxigenic Escherichia coli, on intestinal health parameters can last for up to 21 days after the inoculation, even in the absence of clinical symptoms which ceases around 4 to 11 days 4,9,10 .The change from milk to solid and a less digestible plant-based diet is the major factor altering intestinal microbiota, increasing inflammation, and oxidative damage in the intestinal mucosa 33,35 .When the yeast postbiotic was supplemented in nursery diets, it reduced the fecal score to normal and reduced the incidence of severe diarrhea (pigs with fecal score greater than 5) during the first 7 days post-weaning (Table 1).The reduced fecal score observed in this study might be attributed to increased IFN-γ and mTOR expressions in the postbiotic group.The improved immune responses, indicated by the increased expression of IFN-γ and mTOR, may have led to improved defense mechanisms against pathogenic bacteria, reducing inflammation in jejunal mucosa, although the gene expression of PRR, including TLR2, TLR4, NOD1, NOD2, CD14, and CD3, did not differ between treatments (Table 3).
During inflammation in response to a pathogen, the host produces nitric oxide (NO) as an antimicrobial 36 .The NO is rapidly oxidized to form nitrate that in conjunction with other reactive oxygen species (ROS) causes oxidative stress 37,38 .Under homeostasis, a complex of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), eliminates the ROS reducing the oxidative damages 38 .Excessive ROS generation disrupts the cellular redox balance, leading to the accumulation of oxidative damage products, including protein carbonyls and malondialdehyde (MDA), highly stable markers of oxidative stress 39,40 .Although in this study, the oxidative stress markers were not affected by the treatments, the carbonylation significantly alters protein structure and function, triggering apoptosis pathways 41,42 .
In the current study, the use of yeast postbiotics in a nursery diet reduced the gene expression of BAX1 in jejunal mucosa.BAX1 is a protein that triggers apoptosis by binding and blocking the apoptosis inhibitor BCL2 43,44 .Under stress, BAX1 undergoes a conformational shift, driving its relocation to the mitochondrial membrane.This disrupts the membrane, causing the release of cytochrome c, a key player in initiating programmed cell death that activates caspase-3 (CASP3) 43,45 .Interestingly, the expression of CASP3 was not affected by the treatments in this study, possibly because CASP3 is expressed in its inactive form and is activated when it receives a cell death stimulus 46 .
The reduced expression of BAX1 reported in the current study in pigs fed diets supplemented with yeast postbiotic can be associated with the increased expression of mTOR observed in the same group.mTOR impairs the degradation of myeloid cell leukemia 1 (MCL-1), a protein that prevents apoptosis 47 .However, it is important to note that mTOR can both inhibit and induce apoptosis.mTOR can also induce p53 to promote BAX translocation to the mitochondrial membrane stimulating apoptosis 48 .
The reduced expression of BAX1 and the increased expression of mTOR and IFN-γ seem to have conferred enhanced protection of villi in the jejunum of pigs fed yeast postbiotic.The villus height showed a trend to increase in pigs fed yeast postbiotic.The increased villus height can be a reflection of the increased number of ki67 + cells in jejunal crypt.Ki67 is a marker of cell proliferation associated with activation of mTOR in cancer cells 49,50 .Previous studies using the same product have shown that the yeast-based postbiotic containing bioactive peptides can stimulate growth and differentiation through mTOR activation in muscular cells of pigs 26 and increase crypt depth, an indicator of cell proliferation, in broilers 27 .These results indicate that the yeast postbiotic influences intestinal health by protecting villi by reducing diarrhea and cell apoptosis, increasing cell proliferation and immune responses of the jejunal mucosa.
In conclusion, the supplementation of Saccharomyces yeast postbiotics, at 175 g/ton of diet for nursery pigs reduced the diarrhea incidence within the first week after weaning and provided protection to the villi in the jejunum by enhancing the immune responses of nursery pigs, promoting crypt cell proliferation, and reducing the expression of genes associated with apoptosis without affecting inflammatory and oxidative stress status in the jejunum and the growth of nursery pigs.

Methods
The experimental protocol was approved (Approval number 22-438) by the Institutional Animal Care and Use Committee of North Carolina State University.All methods were performed in accordance with the relevant guidelines and regulations.The experiment is reported in accordance with ARRIVE guidelines (https:// arriv eguid elines.org).

Animals, experimental design, and diets
The experiment was conducted at the Swine Educational Unit at North Carolina State University (Raleigh, NC).Thirty-two newly weaned pigs at 21 d of age (16 barrows and 16 gilts) with initial BW of 6.05 ± 0.24 kg were purchased from a commercial farm (NG Purvis Farms, Robbins, NC, USA) and allotted into two dietary treatments based on a randomized complete block design with initial BW and sex as blocks.The dietary treatments were a control (basal diet) and Postbiotic (basal diet with Saccharomyces yeast postbiotics containing bioactive www.nature.com/scientificreports/peptides at 175 g/ton diet).Saccharomyces yeast postbiotics (celluTEIN) was obtained from Puretein Bioscience LLC (Minneapolis, MN, USA) and are available as a commercial item.The amount of supplementation was previously evaluated by Kim and Duarte 49 .
The Postbiotic was supplemented replacing corn in the basal diet, formulated meeting the nutrient requirements of NRC (2012), as are shown in Table 5. Pigs were housed individually in a pen and had ad libitum access to diets and water.The experimental period was 35 d, which was divided into 3 dietary phases: phase 1 (d 1 to d 10), phase 2 (d 10 to d 19), and phase 3 (d 19 to d 35) to provide nutrients meeting the requirement.Pigs and the diet disappearance were individually weighed by the end of each phase to monitor diet intake and growth of pigs.Fecal score was recorded bi-day from d 3 of experiment.Fecal scores were evaluated daily by the same person considering the following scale: very hard and dry stool (score 1), firm stool (score 2), normal stool (score 3), loose stool (score 4), and watery stool with no shape (score 5) as described by Cheng et al. 50.

Sample collection
After 35 d of the study, pigs were euthanized by exsanguination after a penetrating captive bolt to head.Mucosal samples (from 15 cm, in 3 aliquots) from mid-jejunum (3 m after the duodeno-jejunal junction) were scraped, placed in Eppendorf tubes (2 mL) and later stored at −80 ℃ (after snap-freezing in liquid nitrogen, immediately after collection) for immune and oxidative stress measurements.Jejunal tissues (1 piece, 3 cm) from the midjejunum was collected to measure the expression of gene associated with intestinal barrier markers: Claudins, Occludens, Zonula Occludens-1 (ZO-1), and Mucin 2 (MUC2); as indicators of PRR: TLR2, TLR4, NOD1, NOD2, CD3, and CD14; immune response: NF-κB and IFN-γ; apoptosis and cell proliferation and differentiation: Table 5. Composition of basal diets (as-fed basis). 1 The vitamin premix provided the following per kilogram of complete diet: 6,613.8IU of vitamin A as vitamin A acetate, 992.0 IU of vitamin D3, 19.8 IU of vitamin E, 2.64 mg of vitamin K as menadione sodium bisulfate, 0.03 mg of vitamin B12, 4.63 mg of riboflavin, 18.52 mg of D-pantothenic acid as calcium pantothenate, 24.96 mg of niacin, and 0.07 mg of biotin. 2 The trace mineral premix provided the following per kilogram of complete diet: 4.0 mg of Mn as manganous oxide, 165 mg of Fe as ferrous sulfate, 165 mg of Zn as zinc sulfate, 16.5 mg of Cu as copper sulfate, 0.30 mg of I as ethylenediamine di-hydroiodide, and 0.30 mg of Se as sodium selenite. 3SID, standardized ileal digestible. 4

Oxidative stress and immune status
The concentrations of total protein (Pierce BCA Protein Assay Kit #23227, Thermo Fisher Scientific Inc., Rockford, IL, USA), interleukin 6 (Porcine IL-6 DuoSet ELISA, #DY686, R&D System Inc. Minneapolis, MN, USA), IL-8 (Porcine IL-8/CXCL8 DuoSet ELISA, #DY535, R&D System Inc.), tumor necrosis factor-alpha (Porcine TNF-alpha DuoSet ELISA, #DY690B, R&D System Inc.), protein carbonyl (OxiSelect Protein Carbonyl ELISA Kit, #STA-310, Cell Biolabs, Inc.San Diego, CA, USA), MDA (OxiSelect TBARS Assay Kit, #STA-330, Cell Biolabs, Inc.), immunoglobulin G (Pig IgG ELISA Kit, #E101-104, Bethyl Laboratories, Inc., Montgomery, TX), and IgA (Pig IgA ELISA Kit, #E101-102, Bethyl Laboratories, Inc.) were measured by the colorimetric method using commercially available kits according to instructions of the manufacturers.The absorbance was read using a plate reader (Synergy HT, Biotech Instruments, Winooski, VT) and the software (Gen5 Data Analysis Software, BioTek Instruments).The concentration was calculated based on the standard curve created from concentration and absorbance of the respective standards.All the values were within the respective assay range.The analyte concentrations were normalized using the concentration of total protein within each sample and the data were expressed as unit/mg of protein 51 .

Gene expression of intestinal markers
The RNA extraction from mid-jejunal tissue involved the use of Trizol reagent (Thermo Fisher Scientific Inc.).Subsequently, 1 µg of total RNA underwent complementary DNA synthesis using oligo dT and M-MLV Reverse Transcriptase (Thermo Fisher Scientific Inc.), following manufacturers' instructions.Briefly, quantitative realtime polymerase chain reaction (PCR) was employed to measure relative messenger ribonucleic acid (mRNA) levels, utilizing Applied Biosystems SYBR Green PCR Master Mix (Thermo Fisher Scientific Inc.) and a QS5 Real-Time PCR System.Results were expressed relative to the housekeeping gene β-actin.Importantly, the Ct of the housekeeping gene β-actin remained unaffected by dietary treatment.Normalization of gene expression to β-actin occurred using the delta-delta-Ct method, as previously described 52 , and results were expressed relative to β-actin levels.All primers (Table 6) were rigorously validated for melting curve, efficiency (100% ± 10%), and linearity (r2 ≥ 0.99) of amplification.

Jejunal morphology and crypt cell proliferation
Two sections of fixed mid-jejunum tissues were sent to the Lineberger Comprehensive Cancer Center, School of Medicine at the University of North Carolina (Chapel Hill, NC) to be processed and stained with the ki-67 immunohistochemistry assay following their internal protocol.The slides were used to measure the proportion of proliferating cells in the crypt and jejunal villus height, crypt depth, and villus height to crypt depth ratio (Fig. 1A-D), as previously described by Deng et al. 53 .

Statistical analysis
The experimental unit was pig individually housed and fed.The number of experimental units was pre-determined based on power test 54 .Data were analyzed based on a randomized block design using the Proc Mixed of SAS 9.4 software (SAS Inc., Cary, NC, USA).Dietary treatments were defined as fixed effects and the blocks (sex and initial BW) were the random effects.The data related to diarrhea incidence were analyzed using the Proc Freq of SAS 9.4.The means were calculated using the lsmeans statement in SAS 9.4 and reported with the standard error of the mean (SEM) within each treatment and the SEM related to all values.Statistical differences were considered significant with P < 0.05 and tendency with 0.05 ≤ P < 0.10." Table 6.Sequence of primers for immune responses and barrier function in jejunum.Crypt depth ratio (Fig. 1A-D), as previously described by Deng et al. 53 . 1 Nucleotide oligomerization domain. 2 Mammalian/ mechanistic target of rapamycin. 3Serum and glucocorticoid-induced protein kinase. 4B-cell lymphoma 2-associated X protein. https://doi.org/10.1038/s41598-024-70399-2

Figure 1 .
Figure 1.Representative images of immunohistochemistry (Ki67) staining for jejunal morphology and crypt cell proliferation were obtained.Ten images at 40 × magnification of well-oriented villi and their associated crypts ((A): Control; (B): Postbiotic) were acquired for measuring villus height (from the top to the base of the villus, as indicated with a double arrow line in blue) and crypt depth (from the base of the villus to the bottom of the crypt, as indicated with a double arrow line in red).Ten images at 100 × magnification of the crypts ((C): control; (D): Postbiotic) were captured for counting the Ki67 + staining cells as an indicator of crypt cell proliferation.

Table 2 .
Immune and oxidative stress status of pigs fed diets supplemented with Saccharomyces yeast postbiotics.

Table 3 .
Relative gene expression of intestinal markers in pigs fed diets supplemented with Saccharomyces yeast postbiotics. 1 Mean ± Standard error (n = 8).

Table 4 .
3ejunal morphology of pigs fed diets supplemented with yeast-based postbiotic. 1 Mean ± Standard error (n = 16).2Villusheight to crypt depth ratio.3Number of positive crypt cell proliferation in jejunum.